Biological events usually depend on the cooperative activity of multiple proteins and the precise arrangement of proteins in complexes influences and determines their function. Thus, the ability to arrange individual proteins in a complex in a controlled manner represents a useful tool in characterising protein functions. Moreover, the conjugation of multiple proteins to form a so-called “fusion protein” can result in molecules with useful characteristics. For instance, clustering a single kind of protein often greatly enhances biological signals, e.g. the repeating antigen structures on vaccines. Clustering proteins with different activities can also result in complexes with improved activities, e.g. substrate channelling by enzymes.
However, clustering different kinds of proteins into precise artificial “fusion proteins” has encountered numerous problems. For instance, individual proteins or protein domains can be joined genetically into one long open reading frame, but errors in protein synthesis and misfolding soon become limiting. Alternative methods have focussed on expressing proteins or protein domains individually and then linking these “modules” or “units” together. For instance, methods have focussed on modifying proteins to contain well characterised interaction partners, such as biotin/avidin, thereby enabling proteins to form complexes through non-covalent interactions. Other methods have relied on reactive groups within the proteins, particularly cysteine residues, to link proteins through covalent bonds, i.e. disulfide bridges. However, even the best non-covalent linkages or reversible covalent linkages allow the rearrangement of fusion proteins. Accordingly, existing methods are limited, insofar as they commonly result in undefined mixtures of fusion proteins that are difficult to separate and/or fusion proteins that are not stable across a variety of environments, e.g. in reducing conditions.
Important features of a system for synthesizing fusion proteins include molecularly-defined connections between the individual proteins (i.e. modules, domains or units) within said fusion protein, independence from any template, and simple expression of each protein (i.e. module, domain or unit). It is also highly desirable to have a near quantitative yield for each reaction to minimize the inadvertent synthesis of heterogeneous products after just a few steps, which is a common consequence of incomplete chains within the mixture. It is also preferable for individual modules to be modified with relatively small peptide tags rather than large protein fusion domains, for minimal disruption to the function of each module within the fusion protein. However, no existing fusion protein synthesis method has been able to fulfil these criteria.
Thus, there is a need and desire for an improved method for synthesizing fusion proteins and it has now been found that peptide linkers that form isopeptide bonds to generate irreversible covalent linkages can be used in a modular (e.g. step-wise), and high-yielding, method for synthesizing a fusion protein.